Role of C-mannosylation in the secretion of mindin

• Published in: Biochimica et biophysica acta. General subjects Vol. 1864; no. 8; p. 129632
• Main Authors: Inai, Yoko, Ueda, Kana, Matsui, In-Sook Lee, Tajiri, Michiko, Minakata, Shiho, Wada, Yoshinao, Ihara, Yoshito
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2020
• Subjects: Animals; biosynthesis; C-Mannosylation; calreticulin; Cells, Cultured; Chlorocebus aethiops; complementary DNA; COS Cells; dimethyl sulfoxide; disulfide bonds; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Extracellular Matrix Proteins - metabolism; fluorescent antibody technique; genetic vectors; Glycosylation; immunity; Mannose - metabolism; mass spectrometry; Mice; Mindin; Molecular Chaperones - metabolism; mutants; neurodevelopment; NIH 3T3 Cells; oxidation; Rabbits; Redox; secretion; Thrombospondin type I repeat; DSP; Thrombospondin type I repeat; CRT; 4-PBA; DMSO; Na+/K+-ATPase; Dol-P-Man; AMS; Redox; BiP; Glycosylation; PAGE; PERK; CNX; BSA; DMEM; TSR; C-Mannosylation; PDI; Mindin; TGN46; Endoplasmic reticulum; GAPDH
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129632

Mindin (spondin2), a secretory protein related to neural development and immunity, is a member of thrombospondin type I repeat (TSR) superfamily proteins, and has a unique glycosylation of C-mannosylation in its structure. However, it remains unclear whether C-mannosylation plays a functional role in the biosynthesis of mindin in cells. Protein C-mannosylation was analyzed by mass spectrometry. Mindin expression was examined by immunoblot and immunofluorescence analyses in COS-7 cells transfected with the expression vectors for wild type (mindin-WT) or C-mannosylation-defective mutant of mindin (mindin-mutF). The redox status was examined in mindin by using 4-acetoamide-4′-maleimidylstilbene-2,2′-disulfonate. When mindin cDNA was expressed in COS-7 cells, C-mannosylation of mindin was confirmed at Trp257 by mass spectrometry. In cells expressing a mindin-mutF, secretion of the mutant was significantly inhibited compared with mindin-WT. In immunofluorescence analysis, mindin-mutF was accumulated in the endoplasmic reticulum (ER), whereas mindin-WT was detected in the Golgi. In addition, mindin-mutF showed an enhanced interaction with calreticulin, an ER-resident chaperone, in cells. In cells, reduced forms were increased in mindin-mutF, compared with a mostly oxidized form of mindin-WT. In the presence of chemical chaperones such as dimethylsulfoxide or 4-phenylbutyrate, inhibited secretion of mindin-mutF was ameliorated in cells, although redox-dependent folding was not affected. C-Mannosylation of mindin facilitates its secretion especially through modulating disulfide bond formation in mindin in cells. These results suggest that C-mannosylation plays a functional role in the redox-dependent folding and transport of TSR superfamily proteins in cells. [Display omitted] •C-Mannosylation of mindin controls its secretion.•C-Mannosylation-defective mutant of mindin is accumulated in the ER.•C-Mannosylation facilitates disulfide bond formation in mindin in cells.